Project Summary Post-translational modification of proteins with ubiquitin leads to their degradation by the proteasome. Ubiquitin ligases confer substrate specificity in the ubiquitin-proteasome system by binding target proteins and presenting them for ubiquitination. While thousands of proteins in the cell are ubiquitinated by over 600 ligases, the direct targets of ubiquitin ligases are still difficult to identify. As such, the general principles of substrate recognition remain unclear. Remarkably, a class of small molecules termed IMiDs (immunomodulatory drugs), including thalidomide, lenalidomide and pomalidomide, can bind ubiquitin ligases and alter their substrate specificity by inducing the degradation of novel targets. Recently, another class of compounds (sulfonamides) has been shown to act in an analogous manner by inducing the degradation of the splicing factor RBM39. The long-term goal of the proposal is to understand how small molecules can rewire ubiquitin ligase selectivity from endogenous targets to neo-substrates. The objective of the application is to determine how the sulfonamide compound E7820 alters the target specificity of the ubiquitin ligase substrate receptor DCAF15. The central hypothesis is that E7820 binding to DCAF15 generates a novel small molecule-protein interaction surface that provides specificity for substrates containing an RNA recognition motif (RRM), including RBM39. To address this hypothesis, Aim 1 will utilize a combination of biochemical and structural methods to provide a molecular basis for E7820-mediated recruitment of RBM39 to CRL4DCAF15 for degradation. Visualizing the CRL4DCAF15-E7820-RBM39 complex will reveal how the substrate interaction surface of DCAF15 is repurposed for novel targets. Structure-guided mutants will be generated to support this molecular picture in vivo. Aim 2 will employ a degradation screen of a human RRM-GFP library, which will uncover the full repertoire of E7820- dependent CRL4DCAF15 substrates. Any RRMs stabilized by E7820 treatment are likely natural targets of the CRL4DCAF15 ligase. Insight from these studies will provide a framework for the design of compounds that can alter selectivity of other ubiquitin ligases and facilitate the discovery of natural ligands that similarly regulate ligase-substrate interactions. Finally, targeted protein degradation is an exciting new avenue of therapeutic development, and the proposed work will accelerate the design of new classes of drugs that can degrade a broader range of targets, enabling novel therapies for currently incurable diseases.